Objective T cells are phenotypically categorized into naive precursors(Tn)cells,central memory(Tcm)cells,effector memory(Tem)cells and effector cells.Memory CD8+T cells have a long-lasting ability to quickly respond to antigens.Therefore,these immune cells represent ideal candidates for treating malignancies.During T cell differentiation,transcription factors and microRNAs are dynamically involved in regulating the phenotypic and functional alterations in T cells.Metformin has been widely used to treat type 2 diabetes for nearly 60 years.It also has the potential to improve the survival rate of patients with cancer.Metformin affects multiple cellular pathways via the activation of AMPK by liver kinase 1(LKB1).The activation of the AMPK pathway can restrain mTOR activity,which decreases cell proliferation and induces cancer cell apoptosis.However,the underlying mechanisms involved in improving the memory differentiation of CD8+T cells by metformin are not well defined.Here,we show that metformin regulates memory CD8+T cell differentiation by activating AMPK signaling and up-regulating the expression of Eomesodermin(Eomes).Methods The effect of metformin on overall survival of diabeticpatients with lung cancerwere determined by a Kaplan-Meier analysis.The subsets and function of CD8+T cells derived from both peripheral blood and tumor tissues were analyzed by flow cytometry.Further,the metformin mediated pathway on memory T cell generation were determined by qRT-PCR,western blotting and chip assay.Anti-tumor activity was evaluated after adoptive transfer of the metformin treated HER2 CAR-T cells into HER2+tumor-bearing immunodeficient mice.Results We foundthat patients treated with metformin exhibited a better overall survival(OS)rate than did those who were not(25 vs18 months).The frequencies of memory stem(Tscm,CD45RA+CD62L+CCR7+CD95+)and central memory(Tcm,CD45RA-CD62L+)T cells increased for both peripheral and tumor-infiltrating CD8+T cells in metformintreated lung cancer patients compared to those not taking the medication.In addition,the production levels of IFN-gand IL-2 were enhanced in CD8+T cells from subjectstreated with metformin,and there was no significant difference in the production of TNF-α.Anin vitroassay showed that metformin promoted the formation of memory CD8+T cells and enhanced their anti-apoptotic abilities.Furthermore,AMPactivated protein kinase(AMPK)activation decreased microRNA-107 expression,thereby promoting enhanced Eomes expression and the subsequent suppression of PDCD1 transcription in metformin-treated CD8+T cells.In the CAR-T cell therapy model,the metformin-treated HER2-CAR T cells exhibited a higher percentage of Tscm and Tcm cells than did the control and HER2-CAR T cell groups.After adoptive immunotherapy,the tumor growth in the mouse models was assessed using a bioluminescent(IVIS)imaging system.The HER2-CAR T cells,with or without metformin treatment,strongly suppressed the growth of tumor cells.Importantly,the tumor volume showed a tendency toward better efficacy in the metformin-treated HER2-CAR T cell group than it did in the HER2-CAR T cell group.These data showed that metformin can enhance the anti-tumor abilities of antigen-specific T cells bothin vitroand in vivo.Conclusions Altogether,the data presented in this study showed that metformin promotes the development of memory T cells via the AMPK-miR-107-Eomes pathway.The combination of metformin treatment with T cell therapies provides a promising immunotherapy strategy.